The presence of an abnormally high level of leukocytes in a patient's urine or other bodily fluids is possibly indicative of such pathological conditions as kidney or urogenital tract infection or other dysfunction. Accordingly, accurate urinary leukocyte information can be an invaluable tool to the physician in diagnosis and treatment of such pathologies.
Traditionally, the medical profession has relied on visual determination techniques to count leukocyte population in urine sediment or uncentrifuged urine, a process requiring expensive equipment such as a centrifuge and microscope, as well as inordinate time expenditure on the part of the clinician. Moreover, the traditional techniques suffer from the disadvantage that only intact cells are detected. Leukocytes in the urinary system are subject to conditions which favor extensive cell lysis. For example, it is known that in urines of abnormally high pH, leukocyte half life can be as low as 60 minutes. Since lysed cells escape detection in visual examination techniques, erroneously low determinations and false negatives can result.
In the more recent past, the medical profession has relied on detecting hydrolytic enzymes contained within leukocytes rather than counting the intact cells themselves. Methods for colorimetrically determining the presence of leukocytes through the measurement of hydrolytic esterases and proteases contained within the cells utilize compositions which include chromogenic esters which when hydrolyzed by esterase or protease, produce a colored alcoholic product. Many of these compositions also include accelerator compounds and diazonium salt coupling agents.
Thus, there exists in the prior art a body of references which disclose the use of certain esters which, when cleaved by enzymatic activity, result in the formation of colored or other detectable species. British Patent No. 1,128,371 ('371) discloses the use of indoxyl and thioindoxyl esters as useful chromogens in detecting hydrolytic enzymes in body fluids. The enzymes cleave the ester to generate free indoxyl, which subsequently oxidizes to form the dimeric product indigo, a readily observable blue dye. Such activity is said to be due to, among other enzymes, cholinesterase. The patent '371, also teaches that, in addition to the indoxyl portion of the ester substrate, the acid radical is chosen with particular reference to the enzyme to be detected. For example, it is stated that the acid radical can be acetate, laureate or stearate for detection of esterase or lipase, respectively. For detecting enzymes such as phosphatase or sulfatase the acyl radical can be an inorganic radical. Thus, '371 teaches the use of chromogenic esters as substrates for determining esterolytic enzymes, such esters comprising indoxyl or thioindoxyl as the alcoholic moiety of the ester the acyl moiety being tailored to the particular enzyme to be determined.
The effect of careful acyl radical selection is nowhere more clearly exemplified than in two references which demonstrate esterase specificity for esters in which the acyl radical comprises an N-protected amino acid or peptide. Janoff et al., Proc. Soc. Exper. Biol. Med. 136:1045-1049 (1971), teaches that alanine esters are specific substrates for esterase obtained from human leukocytes. Specifically, Janoff teaches that an extract of human leukocyte granules is capable of hydrolyzing N-acetyl-L-alanyl-L-alanyl-L-alanine methyl ester. Moreover, L-alanine-p-nitrophenyl ester was similarly hydrolyzed to yield the yellow p-nitrophenol colorform.
Similarly, Sweetman et al., Jour. Hist. Soc., 22:327-339, teaches the use of 1-naphthyl N-acetyl-DL-alanine, 1-naphthyl N-acetyl-L-alanyl-L-alanyl-L-alanine and 1-naphthyl butyrate to demonstrate the presence of esterase.
U.S. Pat. No. 4,278,763, assigned to Boehringer Mannheim GmbH, combines these teachings in arriving at the indoxyl or thioindoxyl esters of amino acids or peptides as still another example of a traditional chromogenic substrate for leukocytic esterase activity. Like Janoff and Sweetman, the Boehringer patent teaches the equivalence of protease and esterase in their esterolytic penchants.
It is known that ester hydrolysis reactions can be activated by the presence of many nucleophilic agents, including a myriad of alcohols. Thus, the rate of hydrolysis of phenyl acetate and p-nitrophenyl acetate by esterase is increased 2.5 to 5.5 times upon addition of methanol or butanol. Greenzaid and Jencks, Biochemistry, 10(7), 1210-1222 (1971). Moreover, the effect increases with the length of the n-alkyl group. Wynne and Shalatin, Eur. J. Biochem 31:554-560 (1972).
In particular, this activation effect of alcohols has been observed with esters of amino acids. p-Nitrophenyl-N-acetyl-L-alaninate hydrolysis is activated (accelerated) by the presence of methanol. Fastrez and Fersht, Biochemistry, 12(11), 2025-2034 (1973). High molecular weight alcohols increase the rate of esterase-induced hydrolysis of p-nitrophenyl-t-BOC-L-tyrosinate. Ashe and Zimmerman, Biochem. and Biophys. Res. Comm., 75(1), 194-199 (1977). The disclosure of U.S. Pat. No. 4,299,917 describes other known ester hydrolysis activators such as certain metal complexes, pyridine derivatives and imidazoles.
Also known in the art is the use of certain diazonim salts to couple with phenols and pseudophenols to produce azo dyes. Martinet and Dornier Compt. Rend., 170, 592 (1920). Such a technique is used in an esterase analysis whereby indoxyl esters are hydrolyzed via esterase to produce indoxyl, which is in turn coupled with a diazonium salt to form the corresponding azo dye. Holt and Hicks, J. Cell Biol. 29, 261-366 (1966); Gossrau, Histochemistry, 57, 323-342 (1978); West German Offenlegungschrift No. 30 17 721, filed May 9, 1980.
The diazonium salts known for use as coupling agents in a composition for detecting leukocytes, esterase, or protease rely upon an exogenous anion to counter the diazo cation. Moreover, the formulations discussed thus far each suffers, to at least some extent, from interference or inaccuracy due to the presence of phenolic or other compounds present in the sample which are capable of reacting with the diazonium salt. Such interference can result in false negative assays.
Skjold (U.S. Pat. No. 4,637,979) combined the use of a chromogenic ester. diazonium salt coupling agent, and accelerant into an easy to use dip-and-read test composition and device. In the presence of leukocyte, esterase, or protease, the ester is hydrolyzed to an acid and a phenol. The phenol is then free to couple with the diazonium to produce a color change.
Skjold's composition and test device, however, did not consistently provide accurate results at the basic pHs that most favored enzyme mediated hydrolysis and subsequent coupling of the diazonium with the liberated phenol. Specifically, the color change results were inaccurate at higher pHs because while hydrolysis and diazonium coupling increased with increasing basicity of the reaction mixture, so also did background reactivity of the diazonium with other components in the mixture. Thus, at pHs around 8.8-9.0 color change frequently resulted even in the absence of enzyme. It has been suggested that these background color changes occur with increasing pH of the reaction mixture due to increased nucleophilic attack on the diazonium salt by hydroxide ions. Clearly, there is a need for a reagent composition for detecting leukocytes, esterase or protease which can be utilized at a pH which promotes efficient enzyme mediated hydrolysis and diazo coupling but at the same time promotes diazonium stability in the absence of such enzymes. Additionally, there is a need for a reagent composition in which the diazonium salt remains stable during manufacture.
Diazonium compounds are used in a variety of applications in medicine and industry. Thus, various attempts have been made to try to stabilize such compounds against nucleophilic attack and other types of degradation. Stabilization attempts have included the use of compounds such as organic base, surfactants, organic borates, antioxidants, acid stabilizers, and zinc chloride. None of these approaches was, however, designed to stabilize diazonium salts in a reagent composition formulated to detect leukocytes. Furthermore, none has proven successful in such a composition. Zinc chloride is not effective because it reduces the reactivity of the esterase and protease enzymes. Acid stabilizers do not work because the enzyme detection composition must be basic in order to promote diazonium reactivity and enzyme activity. Surfactants, anti-oxidants, and organic borates were tested but had little effect in stabilizing the diazonium.
Given the limitations of the prior known art, there is a clear need for a method of stabilizing the diazonium against nucleophilic attack and at the same time allow for a composition pH of between 8.8-9.0 in order to promote efficient diazo coupling in the presence of enzyme.